Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions

Abstract

The genes for the alpha and beta chains of a highly reactive anti-MART-1 T-cell receptor were isolated from T-lymphocytes that mediated in vivo regression of tumor in a patient with metastatic melanoma. These genes were cloned and inserted into MSCV-based retroviral vectors. After transduction, greater than 50% gene transfer efficiency was demonstrated in primary T-lymphocytes stimulated by an anti-CD3 antibody. The specificity and biologic activity of TCR gene-transduced T-cells was determined by cytokine production after coculture of T-cells with stimulator cells pulsed with MART-1 peptide. The production of interferon-gamma and granulocyte macrophage-colony stimulating factor (GM-CSF) was comparable to highly active MART-1 specific peripheral blood lymphocytes (PBL) in the amount of cytokine produced and transduced cells recognized peptide pulsed cells at dilutions similar to cytotoxic T lymphocyte (CTL) clones. Human leukocyte antigen (HLA) class I restricted recognition was demonstrated by mobilization of degranulation marker CD107a, by cell lysis, by cytokine production, and by proliferation in the presence of HLA-A2-positive but not HLA-A2-negative melanoma cell lines. Similar data was obtained when tumor-infiltrating lymphocytes (TIL) were transduced with the TCR genes, converting previously nonreactive cells to tumor reactive cells. TCR-transduced T-cells are thus attractive candidates for evaluation in cell transfer therapies of patients with cancer.

Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions

“…The 1G4 ␤-chain TCR was amplified using the ␤1.3F primer that encoded a portion of the P2A sequence followed by sequences corresponding to the beginning of the BV6-5 coding region, and the ␤4.3R primer corresponding to the 3Ј end of the C␤2 C region along with an EcoRI restriction endonuclease site. The two PCR products were then linked in a third PCR conducted with the ␣1.3F and ␤4.3R primers, and the resulting product digested with NcoI and EcoRI and ligated to the MSGV-1 retroviral expression vector which had been digested with the same enzymes (31). For generating the 1G4 ␣95:LY/WT ␤ retroviral variant, the 5Ј ␣-chain fragment was amplified from the native 1G4 WT ␣ construct using ␣1.2F and ␣2.5R primers, and the 3Ј ␣-chain fragment was generated using the ␣3.23F and ␣4.2R primers.…”

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

“…12 The TCRs for a variety of tumor-associated antigens (TAA) have been identified including the MART-1 and gp100 melanoma differentiation antigens, the NY-ESO-1 cancer-testis antigen and the p53 tumor suppressor. [13][14][15][16][17][18] An important aspect of TCR expression vector design is that, the TCR-a-and -b chains must be coordinately expressed for proper biologically activity. 12 The most common strategy for the expression of multiple genes has been based on internal ribosome entry site (IRES) elements.…”

A Novel Fusion of ALT-803 (Interleukin (IL)-15 Superagonist) with an Antibody Demonstrates Antigen-specific Antitumor Responses

“…Similar to the sc2B8 fusions, the c264scTCR was fused to IL-15N72D or IL-15R␣SuFc to make the c264scTCR-IL-15N72D and c264scTCR-IL-15R␣SuFc constructs. The resulting sc2B8-IL-15N72D⅐sc2B8-IL-15R␣SuFc (2B8T2M), sc2B8-IL-15D8N⅐sc2B8-IL-15R␣SuFc (2B8T2M-D8N), sc2B8-IL-15N72D⅐sc2B8-IL-15R␣SuFc-LA (2B8T2M-LA), and c264scTCR-IL-15N72D⅐c264scTCR-IL-15R␣SuFc (c264T2M) genes were expressed in the pMSGV retroviral vector (28).…”